Urinary tract infections, primarily cystitis account for over six million physician office visits a year in the United States alone. The colonic organism E.Coli accounts for the majority of cases, following an ascending route from the periurethral area into the bladder. The resulting infection can range from an asymptomatic bacteriuria (ABU) to cystitis, pyelonephritis or urosepsis. Cytotoxic Necrotizing Factor 1 (CNF1) is an E. Coli toxin which is linked epidemiologically to UTI and which alters the cytoskeleton, morphology, and division of toxin treated cells in vitro. Moreover, the role of CNF1 in pathogenesis is unknown. CNF1 biochemically modifies the small GTPase Rho which regulates the formation of focal adhesions to substrata and proliferation of animal cells in vitro. Since these are critical steps in the finely tuned processes of epithelial development, we hypothesize that CNF1 contributes to pathogenesis by interfering with the regeneration of damaged epithelium. Using clinical isolates of E. Coli, bacterial lysates containing CNF1, and in vitro models of migration, proliferation, and wound repair, we will determine effects of this toxin on the target human bladder epithelial cell. Additional E. Coli genes required for the delivery and action of CNF1 will be identified by transposon mutagenesis.